During the first funding period of this grant our goal was to develop the humanized BLT mouse system as a highly innovative model for the evaluation of different topical microbicides and systemic drugs for HIV prevention. We succeeded in this endeavor in ways that go beyond our initial proposal, published 11 papers and the BLT mouse model in now recognized as an excellent model for the pre-clinical evaluation of virtually all types of HIV prevention approaches. However, also during the tenure of this grant results from several large clinical trials of HIV prevention have brought to the forefront significant problems concerning compliance with both topical microbicides and systemic drug therapy. Indeed, the results from human clinical trials show that drugs like tenofovir when used by high-risk individuals are highly effective at preventing HIV transmission, but lack of compliance has been correlated with lack of protection. Therefore the focus of the prevention field has now shifted to alternatives that can improve compliance and thus reduce HIV transmission. One approach has recently gained significant attention: long-acting (LA) injectable formulations. Having completed the development and initial testing of the BLT model for the evaluation HIV preventive strategies and in an effort to continue to be avant-garde, in thi competitive renewal application we are addressing the critical issue of compliance by proposing the in vivo evaluation of a LA injectable formulation of Rilpivirine as a proof-of- concept for thi approach. LA antiretroviral preparations that stably release drugs over many weeks as nano-formulations are a novel approach to pre-exposure prophylaxis. This approach offers major benefits including: (a) the ability to mitigate poor patient compliance with daily systemic PrEP dosing; (b) the potential for a reduced reliance on frequently used HIV therapeutic drugs; and (c) their ability to be utilized discreetly without requiring a partner's knowledge or consent. To date no long-acting drug formulations have been evaluated for HIV prevention efficacy as systemic PrEP, which leads to the fundamental question of this proposal: Can long- acting PrEP provide durable protection from HIV transmission? Several drugs are being formulated for sustained release including Rilpivirine (RPV; TMC278), a recently approved non-nucleoside reverse transcriptase inhibitor (NNRTI). RPV was identified as an excellent antiretroviral candidate for LA formulation because it interferes early in the HIV life cycle, is effective against a broad rang of wild type and mutant viruses with reduced susceptibility to commonly prescribed agents, and has low cross-resistance to other NNRTIs. In addition, it has a better tolerability profile than other similar drugs and has fewer neuropsychiatric and metabolic side effects than efavirenz. This depot formulation of Rilpivirine, RPV-LA kindly provided to us by Janssen R&D, has several characteristics that make it a quality candidate for long-acting systemic PrEP. Intramuscular injection of RPV-LA in humans was found to be safe, had no side effects and resulted in sustained plasma and cervical-vaginal fluid drug concentrations for at least 12 weeks. In addition, there were no changes in QT interval. The ability of RPV-LA to prevent vaginal HIV transmission will be systematically tested in the state-of-the-art bone marrow, liver, thymus (or BLT) humanized mouse model that has become widely utilized in human immunology and infectious disease research. For this purpose we will utilize highly relevant transmitted/founder viruses from a variety of HIV subtypes responsible for a significant portion of the AIDS epidemic. Specific Aim 1) To perform pharmacokinetic analyses of RPV-LA in mice to determine RPV concentrations and biodistribution over time with a focus on the duration of drug exposure plasma and in the female reproductive tract (FRT). Specific Aim 2) To perform pharmacokinetic-pharmacodynamic (PK-PD) analyses directly relating RPV concentrations with the efficacy of RPV-LA for preventing vaginal HIV transmission in BLT mice. Specific Aim 3) To evaluate the protective effect of systemic PrEP with RPV-LA from acquisition of naturally occurring, highly relevant transmitted/founder HIV isolates.